Antibodies are well known to bind antigens and it is generally recognized that the antigen-binding segment of antibodies is composed of the variable portion of a heavy (H) and a light (L) chain. Both of these chains are thought to be important in defining the paratope conformation to one that binds antigen with high affinity. It has recently been found that antibodies can catalytically enhance the rate of chemical reactions. In U.S. Pat. No. 4,488,281, it is disclosed that catalytic antibodies can bind a substrate, cause the conversion thereof to one or more products, and release the product. The catalytic antibodies may be prepared by immunological methods wherein they are elicited to antigens, as taught, for example, in U.S. Pat. No. 4,888,281.
Fab fragments of an antibody catalyze hydrolysis of an amide bond (1). Fv fragments, which are heterodimers consisting of the variable regions of associated light and heavy chains of an antibody, have been shown to catalyze ester hydrolysis (2). These antibody components are not known to catalyze the cleavage or formation of peptide bonds, a class of reactions which is energetically more demanding.
Iverson and Lerner (3) report that while peptide bond cleavage is very energetically demanding, cleavage of a peptide bond by a catalytic antibody is enabled by the presence of a metal trien cofactor and will not take place without the presence of such a cofactor. The trien complexes of Zn(II), Ga(III), Fe(III), In(III), Cu(II) , Ni(II), Lu(III) , Mg(II) or Mn(II) were most favored. However, a naturally occurring autoantibody able to selectively catalyze the cleavage of the peptide bond between amino acid residues 16 and 17 of the neurotransmitter vasoactive intestinal peptide (VIP) without any metal cofactor, has been reported by Paul (4, 5).
It is also known that antibody binding is energetically most favored by the presence of the entire H-chain and L-chain binding site (6). The V.sub.H fragments of anti-lysozyme antibodies bind the antigen with an affinity of only 10% of the intact antibody (7). L-chains are also likely to participate in antigen binding interactions, although most studies suggest that the contribution of L-chains is smaller than that of H-chains (8-10). It could not be expected that an antibody component smaller than an intact catalytic antibody would possess the favorable steric conformation provided by the intact catalytic antibody to permit the catalysis of a peptide bond without the assistance of a metal trien cofactor as taught by Lerner and Iverson.
The reports of Fv and Fab catalysis of ester and amide bonds do not disclose that other types of heterodimers catalyze any chemical reactions (8-10). A heterodimer not known or expected to catalyze chemical reactions is the heterodimer consisting of an intact H-chain and intact L-chain linked by at least one disulfide (S--S) bond. Another heterodimer not known or expected to catalyze chemical reactions is a heterodimer analogous to the Fab, but consisting of the Fd- fragment (the H-chain with the Fc portion removed) linked to or associated with an intact L-chain by non-covalent bonding (e.g. hydrogen bonding, charge interaction or similar association), in contrast to the Fab which consists of the Fd fragment linked to the L-chain by at least one disulfide bond.
Heavy chain homodimers and light chain homodimers have heretofore not been shown to catalyze chemical reactions. It would not be expected that these homodimers would have catalytic activity because the classic binding function of antibodies is considered to require the combination of the variable regions of both a light and heavy chain, or at least a heavy chain (8, 11-13). Catalytic light and heavy chain homodimers would be advantageous because they consist of the same or similar components, and thus could be manufactured with less effort than is required to manufacture a standard antibody or a heterodimer.
There are obvious advantages that single chain proteins offer over multichain proteins (antibodies), both from the point of view of structure-function analysis as well as pharmacological and therapeutic stability. It would be advantageous if the binding and catalytic domains on an antibody were either the same or closely positioned to one another such that the benefits of catalytic activity could be achieved by a simple protein as opposed to a multichain antibody. Heretofore, the art has not demonstrated the capability of using such components of an antibody for catalytic purposes. Similar advantages are offered by dimers formed of the several combinations of light and heavy chains.
It is known to use a catalytic antibody to convert a prodrug to a drug (14). However, a catalytic component able to convert a prodrug to a drug, or a protoxin to a toxin has special advantages, particularly when the catalytic component is incorporated into a fusion or chimeric protein with a biological binding agent able to bind to cells or tissues which it is desirable to contact with the drug or toxin.